This document discusses the principle and applications of enzyme-linked immunosorbent assay (ELISA). ELISA uses an enzyme-linked antibody or antigen to detect a protein. It has advantages over radioimmunoassay like being non-radioactive, more stable reagents, and ability to automate. ELISA is used to detect hormones, tumor markers, antigens, and antibodies involved in infectious diseases. Variations include indirect ELISA for antibody detection, sandwich ELISA for antigen detection, and competitive ELISA. The document also discusses other immunoassay techniques like immunofluorescence, immunohistochemistry, EMIT, and lab-on-chip technologies.

1. Enzyme Linked Immunosorbent Assay (ELISA ) and
its clinical applications
Dr. Rohini C Sane

2. Principle of Enzyme Linked Immunosorbent Assay—(ELISA )
Principle of Enzyme Linked Immunosorbent Assay (ELISA ) is as
follows :
• ELISA is based on the immunochemical principle of antigen antibody
reactions .
• It is based on the specificity of antigen antibody complex formation
and its detection by a second antibody conjugated with suitable
enzyme such as peroxidase.

3. Principle of Enzyme Linked Immunosorbent Assay(ELISA)

4. Technique of ELISA
• The technique of Elisa is similar to in principle to that of
radioimmunoassay except the antigen is labelled with a stable
enzyme* instead of a radioisotope compound.

5. Enzymes of ELISA
Enzyme* Source Specific Enzyme
activity (units/mg)
Alkaline phosphatase Calf intestine 400
Beta Galactosidase E.coli 400
Glucose oxidase Aspergillus Niger 200
Glucose -6-phosphate
dehydrogenase
Leucon Stoc
mesenteroides
250
Peroxidase Horseradish 900
 A unit of Enzyme activity represents the conversion of 1 mol of Enzyme substrate to product
per minute .

6. Technique of ELISA
1. The antibody(e.g. anti- HCG ) against the antigen (e.g. HCG-human chorionic gonadotropin ) to
be determined is fixed on an inert solid such as polystyrene ( multi well titer plates ) .
2. The biological sample (e.g. serum ) containing the protein /antigen (e.g. HCG ) is applied on
antibody coated surface.
3. The antibody (e.g. anti- HCG )binds only to antigen (e.g. HCG )from serum. Other proteins get
washed off.
4. The second protein specific antibody conjugated with enzyme (e.g. Horse Radish Peroxidase –
HRP) is added. This binds to antigen (e.g. HCG )forming a complex sticking to plastic surface .
5. Enzyme (e.g. Horse Radish Peroxidase –HRP) is covalently linked to second protein specific
antibody. The enzyme must be assayable & products must be preferably colored product.
Horseradish Peroxidase ,Amylase & Alkaline phosphatase can be commonly used .
6. Excess antibody – Peroxidase is washed off. The amount of antibody – Peroxidase conjugate
sticking to antibody coated surface is proportional to the amount of the antigen (e.g. HCG-
human chorionic gonadotropin ) and is then determined /assayed .
7. This is assayed by adding substrate ( tetra methyl benzidine ). Peroxidase converts substrate to
color product.
8. The color intensity is measured by spectrophotometer .

7. Diagrammatic representation ELISA assay

8. Steps of ELISA

9. Polystyrene ( multi well titer plates ) for ELISA

10. Color complex formation in ELISA
H2O2 + Peroxidase H2O + (O) Nascent Oxygen
(O) Nascent Oxygen + Diaminobenzidine- DAB (colorless)  oxidized
Diaminobenzidine (brown)
Tetra methyl benzidine can be used instead of Diaminobenzidine.
The color intensity of product is proportional to the antigen in the serum .
The intensity of colored complex is measured by spectrophotometer , from
which the concentration of antigen is calculated.
Other chromogens used in ELISA test are NBT ( Nitro Blue Tetra Azolium –
blue color ) , NPP – para nitro phenyl phosphate  (yellow color)

11. Color complex formation in ELISA
Polystyrene ( multi well titer plates ) of ELISA microplates after color complex formation in assay .

12. ELISA Reader

13. Types of ELISA
❖Types of Elisa are as follows:
1. Indirect technique to detect antibody
2. Double antibody technique to detect antigen-Sandwich method
3. Competitive ELISA-single antibody method

14. Types of ELISA
1. Indirect technique ( to detect antibody)
2. Double antibody technique (to detect antigen)-Sandwich method

15. 1. Indirect technique of ELISA ( to detect antibody)
Diagnosis of AIDS ( detection of Human immunodeficiency virus - HIV antibody )
Antibodies from Patient's
serum
HRP
Diaminobenzidine + H2O2 Color intensity directly proportional to the antibody concentration

16. 1. Indirect technique of ELISA ( to detect antibody)

17. 2. Double antibody technique of ELISA (to detect antigen)
❖Principle of Double antibody technique (Sandwich assay) of ELISA :
• On the surface of test tube (or microtiter plate )specific antibody(e.g. anti T4) is coated.
• Serum (the antigen which is to be analyzed-e.g. Thyroid hormone T4) is added in the well. After
mixing it is kept for incubation at 37º C. The antigen (e.g. Thyroid hormone T4) present in serum is
fixed to antibody.
• The micro well is washed to get rid of excess antigens and other unwanted proteins .
• Enzyme( linked specific second antibody (e.g. antibody against T4 tagged with ENZYME-HRP ) is
added & kept for incubation at 37º C.
• Enzyme (e.g. Horse Radish Peroxidase –HRP) linked specific second antibody combines with
already bound antigen forming antibody antigen antibody complex .Thus antigen is sandwiched
between two antibodies .
• After washing off the excess of antibodies ,the enzyme substrate(Diaminobenzidine + H2O2) is
added .
• Enzyme(HRP) acts on substrate & at the end of reaction a color complex is formed .
• If enzyme is alkaline phosphatase the substrate is para nitro-phenyl phosphate (PNPP)
Para Nitrophenyl Phosphate (PNPP)+ Alkaline phosphatase Para Nitro Phenol (Yellow color
complex)
• The enzyme activity is measured by measuring color intensity of product using
spectrophotometer . The enzyme activity is directly proportional to the amount of attached
antibody & antigen present in a test sample .

18. Sandwich ELISA assay (to detect antigen)

19. Sandwich ELISA assay (to detect antigen)

20. 3. Competitive Elisa-Single Antibody Method
2.Double antibody technique (to detect antigen)-Sandwich method
1.Indirect technique ( to detect antibody)

21. 3. Competitive Elisa-Single Antibody Method
In Competitive Elisa-Single Antibody Method
• Known amount of enzyme labelled antigen and unknown amount of
unlabeled antigen from patient sample ,is allowed to react with a specific
antibody attached to a solid phase.
• There is a competition between the labelled antigen and unlabeled antigen
for binding with limited number of antibodies .
• After specific time of incubation ,any unbound antigens are washed off
with buffer.
• Then enzyme substrate is added and enzyme activity is measured by
measuring intensity of colored product formed. (using a
spectrophotometer )
• The enzyme activity is inversely proportional to the amount of unlabeled
antigen from patient sample.

22. 3.Competitive ELISA-Single Antibody Method

23. Different types of Elisa formats

24. DirectELISA for HIV antigen detection
HIV antigen

25. Signal amplification by Biotin –Avidin Complex in ELISA
• Biotin will tightly bind with avidin .
• Instead of enzyme directly fixed over antibody ,biotin is labelled on the
first antibody. The avidin conjugated enzyme is added, & color
reaction is done as before .
• The advantage here is that for each biotin fixed ,4 avidin molecules ,&
so 4 enzyme molecules are fixed .
• The intensity of color the assay is thus increased many times .

26. Signal amplification by Biotin –Avidin Complex in ELISA

27. Signal amplification by Biotin –Avidin Complex in ELISA

28. Advantages of Enzyme Linked Immunosorbent Assay(ELISA )
Advantages of Enzyme Linked Immunosorbent Assay(ELISA ) include :
1. Specific & sensitive assay method
2. Small amount of specimen & single dilution is required to perform the
test.
3. The reagents are stable& have longer half life than those used in RIA.
4. The results can be read visually.
5. Large number of specimens can be tested at a time.
6. Elisa can be automated.
7. Non-isotopic assay-enzyme is used in place of radioactive isotope.
( employed in RIA )
8. No risk of radio active hazards in ELISA. ( As in case of with RIA )
9. Elisa is cheaper than RIA (suitable for use even in small laboratories ).

29. Applications of Enzyme Linked Immunosorbent Assay (ELISA )
Applications of Enzyme Linked Immunosorbent Assay (Elisa ) include:
• Estimations of hormones (e.g. LH, FSH, Prolactin )
• Estimations of Tumor markers( e.g. PSA,AFP,HCG ,CEA )
• Estimations of bacterial (hepatitis B surface antigen )/fungal /viral antigens in
biological fluids .
• Test is commonly employed to detect /measure antibodies (present in small
quantities in tissue /blood ) in infectious diseases ( e.g. auto antibodies- anti-
DNA,ANA –antinuclear antibody/ antibacterial antibodies/ antiviral antibodies )
• Diagnosis of AIDS ( detection of Human immunodeficiency virus - HIV antibody )
• The most commonly used pregnancy test for the detection of human chorionic
gonadotropin (beta HCG) in urine is based on ELISA .
Pregnancy can be detected within few days after conception using ELISA.

30. In ELISA the results can be read visually (e.g. pregnancy test)

31. Immuno-fluorescence
❖Instead of enzyme labels fluorescent immunoassay (FIA) or
chemiluminescent immunoassay (CLIA) may be used for
qualitative or quantitative analysis .
• Antibody tagged with fluorescein isothiocyanate is incubated with
cells.
• Antibody fixes with cell surface antigens .
• Subpopulation of cells ( e.g. helper T cells ) are enumerated by this
technique.

32. Direct and indirect Immuno-fluorescence

33. Immunocytochemistry
• Histopathology sections are layered with antibody tagged with Horse
Radish Peroxidase –HRP and then hydrogen peroxide + chromogen
are added. Color develops if antigens are present.
• Slide having tissue sample from cancer (e.g. colon cancer) is reacted
with specific antibody against an oncogene. Color develops wherever
oncogene is present.

34. Immunocytochemistry

35. Immunohistochemistry
Immunohistochemical staining of Human endometrium showing strong nuclear positivity in glandular and

36. Direct and indirect Immunostaining

37. Immunohistochemistry verses Immuno-fluorescence

38. Enzyme multiplied Immunoassay (EMIT)
• In ELISA ,antibody is tagged with enzymes ,but in EMIT antigen is
labeled with enzymes. Serum containing antigen and antibody
are reacted. When antigen-antibody complex is formed. The active
site of enzyme is not available for substrate binding .Such a
system will eliminate the separation of antigen-antibody complex
or the washing procedure .
• This is a definite advantage of EMIT over RIA and ELISA
technique .
• So EMIT is more suitable for automated machines .

39. Principle of Enzyme multiplied Immunoassay (EMIT)

40. Enzyme multiplied Immunoassay (EMIT)

41. Therapeuticapplicationof Enzyme multiplied Immunoassay(EMIT)

42. Lab on chip technology

43. Immunoassays based on lab on chip technology
4 possible immunoassays binding configuration suitable for biosensing application in lab on chip technology

44. Integrated CNT photodetector on lab on chip technology
Immunoassays in microfluidic channel with integrated CNT photodetector

45. Disposable polymer in lab on chip technology for Immunoassays
Immunoassays in microfluidic channel with integrated CNT photodetector